The gene therapy revolution in ophthalmology

Saudi Journal of Ophthalmology (2013) 27, 107–111

Review Article

The gene therapy revolution in ophthalmology Fahad I. Al-Saikhan, Pharm. D ⇑

Abstract The advances in gene therapy hold significant promise for the treatment of ophthalmic conditions. Several studies using animal models have been published. Animal models on retinitis pigmentosa, Leber’s Congenital Amaurosis (LCA), and Stargardt disease have involved the use of adeno-associated virus (AAV) to deliver functional genes into mice and canines. Mice models have been used to show that a mutation in cGMP phosphodiesterase that results in retinitis pigmentosa can be corrected using rAAV vectors. Additionally, rAAV vectors have been successfully used to deliver ribozyme into mice with a subsequent improvement in autosomal dominant retinitis pigmentosa. By using dog models, researchers have made progress in studying X-linked retinitis pigmentosa which results from a RPGR gene mutation. Mouse and canine models have also been used in the study of LCA. The widely studied form of LCA is LCA2, resulting from a mutation in the gene RPE65. Mice and canines that were injected with normal copies of RPE65 gene showed signs such as improved retinal pigment epithelium transduction, visual acuity, and functional recovery. Studies on Stargardt disease have shown that mutations in the ABCA4 gene can be corrected with AAV vectors, or nanoparticles. Gene therapy for the treatment of red–green color blindness was successful in squirrel monkeys. Plans are at an advanced stage to begin clinical trials. Researchers have also proved that CD59 can be used with AMD. Gene therapy is also able to treat primary open angle glaucoma (POAG) in animal models, and studies show it is economically viable. Keywords: Gene therapy, Vector, Retinitis pigmentosa, Leber’s Congenital Amaurosis (LCA), Stargardt’s ophthalmology, Age related macular degeneration (AMD), CD59, Primary open angle glaucoma (POAG) Ó 2013 Saudi Ophthalmological Society, King Saud University. All rights reserved. http://dx.doi.org/10.1016/j.sjopt.2013.02.001

Introduction Gene therapy refers to the use of genes to treat various medical conditions. The concept involves transferring genetic material into cells, tissues or organs with the aim of curing a disease or improving the clinical status of a patient. The central goal of gene therapy is to replace genes that are nonfunctional or defective with new genes that are fully-functional so that the level of genetic expression can return to normal.1 Some of the latest breakthroughs in the field of gene therapy have been its potential applications in the treatment of a wide range of ophthalmic conditions. Most

of the work has focused on the retina which has been shown to be an ideal target especially for the adeno-associated viral (AAV) vector (What is gene therapy? 2012). AAV is a vector that infects the retinal cells quite effectively (Fig. 1). Injection of AAV under the retina entails vitrectomy, which is a standard surgical procedure. After vitrectomy, a very narrow needle is used to inject the vector under the retina (Fig. 2). The AAV is contained in approximately 1/10th of a milliliter of fluid.2 The need for gene therapy is based on the clinical characteristics of an ophthalmic condition. The Gene Therapy Center facilitates both genetic research and gene therapy. Gene therapy trials require approval by the Food and Drug

Received 15 September 2012; received in revised form 6 November 2012; accepted 4 February 2013; available online 11 February 2013. College of Pharmacy, Salman Bin Abdulaziz University, Al-Kharj, Saudi Arabia Department of Pharmacy Services, King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia ⇑ Corresponding author. Address: Assistant Professor & Vice dean for Academic Affairs, College of Pharmacy, Salman Bin Abdulaziz University. P.O. Box 173, Al-Kharj 11942, Saudi Arabia. Tel.: +966 1 4821234x3121; fax: +966 4816226. e-mail address: [email protected] Peer review under responsibility of Saudi Ophthalmological Society, King Saud University

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Figure 1. Image showing how AAV vector is used to mediate gene transfer. (Source: see Ref. 9).

Figure 2. Image showing how DNA or RNA is introduced into the desired site in the retina during gene therapy. (Source: see Ref. 12).

Administration (FDA). Trials that receive funds from the National Institutes of Health (NIH) must be recognized by the NIH Recombinant DNA Advisory Committee (RAC).3 Given the many success stories in the application of gene therapy in ophthalmology, this paper shall give a review of the ongoing research in this area with special reference to six ophthalmic conditions; retinitis pigmentosa, Leber’s Congenital Amaurosis (LCA), Stargardt disease, red–green color blindness, age related macular degeneration (AMD), and primary open angle glaucoma (POAG).

Gene therapy for the treatment of ophthalmic conditions Retinitis pigmentosa (RP) Retinitis pigmentosa (RP) belongs to a group of eye diseases caused by multiple mutations in the gene that encodes rhodopsin. Rhodopsin is an essential pigment that facilitates

the cascade for visual transduction. Visual transduction is the process that enables vision in bright and poorly-lit conditions.4 A mutation in the rhodopsin gene results in a progressive depletion of photoreceptor cells that are responsible for changing light energy into nerve signals. The progressive breakdown of these cells is accompanied by a gradual loss of vision which may initially start with night blindness and progress to total blindness.5 Both rods and cones are photoreceptor cells. Rods are active during mesopic and scotopic conditions whereas cones are active during photopic conditions and for color detection.6 As RP occurs due to gene mutations, researchers have concentrated on using gene therapy to correct the defective genes. Two approaches have been used; the first approach is to transfer a properly functioning copy of the affected gene using AAV into the retina. Alternatively, researchers can inactivate a mutated gene responsible for the production of a gene product that has deleterious effects on photoreceptors.7 Significant success has been achieved by using AAV to mediate transgene expression in the retinal tissue i.e. the

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The gene therapy revolution in ophthalmology pigmented and photoreceptor epithelial cell layers. The ability of AAV to facilitate the transfer of reporter genes to the retina has paved way for recombinant adeno-associated viral (rAAV) vectors in the treatment of retinal degeneration (Fig. 1).8 Nagatsu et al.8 conducted tests on the potential application of rAAV vectors in the treatment of recessive RP due to a mutation in cGMP phosphodiesterase (PDE). They found that after intraocular delivery of the correct PDE into a mouse model that was PDE recessive, photoreceptor cells were protected from degeneration while retinal function was preserved.8 Autosomal dominant RP (ADRP) is another form of RP in which AAV vectors have been shown to have a remarkable therapeutic potential. ADRP is caused by a defective rhodopsin gene product that leads to photoreceptor cells’ death which eventually leads to blindness.10 Nagatsu et al.8 showed that rAAV vectors can deliver ribozyme specifically designed to cut and destroy the mRNA of the defective rhodopsin gene. By using a transgenic rat model of ADRP, Lewin et al. noted reduced degeneration of photoreceptor cells following the delivery of the ribozyme.8 In a recent study, researchers investigated the potential of gene therapy in the treatment of X-linked RP (XLRP). The most prevalent form of XLRP results from a RP GTPase Regulator (RPGR) gene mutation, found in the X chromosome (Beltran et al., 2012). In dogs, this disorder is known as X-linked progressive retinal atrophy (XLPRA), which also emanates from a RPGR gene mutation. By using dog models, researchers used AAV vectors to inject one eye of the experimental dogs with a normal RPGR gene from humans. The solution containing the vector was targeted at the space between the retinal pigment epithelial layer and the photoreceptor cell layer (Fig. 2).11 The other eye was not injected with AAV vector solution and thus served as a control.11 The eyes that had received AAV vector solution showed a resumption of normal RPGR gene expression in the photoreceptors. In addition, the treated areas showed no further retinal degeneration. Retinal thickness was near normal limits in the treated region while the retina in untreated regions became thinner.11 This study provided a firm foundation for the application of a similar approach in humans.

Leber’s Congenital Amaurosis (LCA) LCA is an autosomal recessive disease resulting from mutations in different genes, 15 of which have been reported to date.13 Patients with LCA manifest with fundus abnormality on ophthalmoscopy, impaired pupillary light reflexes, severely reduced electroretinograms, and nystagmus.14 Additionally there is progressively degenerating cellular structure of the retina and severe visual loss and retinal functions. Many studies have investigated the role of gene therapy for the treatment of LCA type 2 (LCA2). LCA2 is associated with mutations in the RPE65 gene (S, L and K 2011). Mutation in this gene causes a deficiency retinoid isomerohydrolase, which facilitates proper functioning of photoreceptors.15 Canine, porcine and rodent models have been used to show that rAAV can deliver functional retinoid isomerohydrolase with the goal of preventing retinal degeneration and restoring visual function.16 The outcomes of this preclinical trial16 has led to a cooperative effort by Pennsylvania University and Applied Genetic Technologies to develop a rAAV

vector with the capacity to express human retinoid isomerohydrolase with a chicken beta-actin promoter being used to control expression. Following the administration of rAAV2CBS-hRPE65 in young adult pigs with LCA2, there was improved visual function in the long term with no indication of an adverse immunogenic response.16 rAAV vectors carrying RPE65 cDNA have been used to treat mice with Rpe65 gene mutation at different stages of development. Cai et al.17 found that treatment outcomes irrespective of the stage of development indicated efficient retinal pigment epithelium (RPE) transduction.17 In addition, the expression of the injected RPE65 protein could be detected even 7 months after injection via immunohistochemistry.17 The treated mice also showed nearly normal retinal morphology and almost normal levels of retinyl-esters and rhodopsin.17 Visual acuity also improved, and remarkable functional rescue was also noted.17 Swedish Briard dog models have also been used to demonstrate the potential therapeutic implications of gene replacement therapy in LCA2.17 In a study by Cai et al.17 several dogs at different ages were each administered with a single injection of rAAV carrying functional RPE65 gene. The results indicated significant improvement in visual function, visual behavior, and retinoid content.17 Two weeks following treatment, functional recovery was clearly evident and peaked 3 months after the injection.17 Functional rescue went beyond 7 years in treated dogs.17 The success seen in animal studies has prompted a team of scientists in London to initiate the first gene therapy trial on humans with LCA. Twelve people with impending blindness due to LCA underwent injections of rAAV vectors carrying the wildtype RPE65 gene into their retinal cells. The physicians also injected normal copies of RPE65 gene directly into the cells of the retina and noted production of wildtype RPE65 proteins.18 There is, therefore, much hope that gene therapy will work in humans with LCA at some point.

Stargardt disease Stargardt disease causes retinal degeneration following mutations in a gene called ATP-binding cassette sub-family A member 4 (ABCA4; ABCR). The gene ABCA4 encodes a protein responsible for transportation of energy between photoreceptor cells and the retina. A mutation therefore, causes the degeneration of photoreceptor cells, which eventually leads to vision loss.19 A number of rodent models mostly using mice have been conducted and promising results have been accumulated.20 For example, Dr. Muna Naash on August 17, 2012 used nanoparticles to transfer normal copies of ABCA4; ABCR gene into mice. This process resulted in long-term preservation of vision in mice with Stargardt disease.21 The outcomes from mice models lay the foundation for further research in how similar approaches can be used to treat Stargardt disease in humans. Nanoparticles are advantageous due to the lower likelihood of precipitating an immune response because of the smaller size compared to human engineered viral vectors.22

Red-green colorblindness Gene therapy to cure red–green color blindness is in advance stage because of success in treating squirrel mon-

110 keys.23 Red–green color blindness affects one out of twelve men and one out of two hundred and thirty women.23 Squirrel monkeys are preferred in this experiment because they are naturally red–green color blind. Researchers at the University of Washington, Department of Ophthalmology proved that gene therapy is able to cure red–green color blindness in squirrel monkeys. The treatment of the disorder entailed injecting the monkeys with a virus that had been modified to deliver corrective gene to the monkeys. However, researchers have faced numerous challenges in their bid to use the therapy to treat red–green color blindness in humans. These challenges include legal hurdles, and financial challenges. Hence, it is pivotal for industrial partners to invest in projects to achieve success. Therefore, clinical trials for therapy are in the planning stage.23 The success of the therapy raised questions about fundamental assumptions about neural development and mental perception because it was believed that this therapy could only be successful in young specimens.

Age related macular degeneration Age related macular degeneration (AMD) is a chief cause of blindness in the elderly, particularly those over 60 years old.24 Gene therapy using CD59 seems to be capable of slowing the progression of AMD. Researchers from Tufts University School of Medicine proved that CD59 administered through gene therapy caused a significant reduction of uncontrolled blood vessel growth as well as dead cells that cause AMD.24 AMD is caused by an activation of membrane attack complex (MAC), which kills cells in the back of the eye, causing AMD. CD59 reduces the development of MAC. The researchers found that CD59 could be delivered to the eye of the patient using a virus that is similar to one used in humans. The findings show that CD59 reduced MAC development by 62%.24 Another implication in the treatment of AMD is that unlike the current mode of treatment, where the patient undergoes frequent injections directly in the eye, gene therapy is less frequent therefore reducing patient discomfort and the risk of infections. The researchers have not performed clinical trials on the use of CD59 in treating AMD. However, they have formed a partnership with Hamera Biosciences, Inc., in a bid to get the required funding.24

Primary open angle glaucoma (POAG) Primary open angle glaucoma (POAG) is a major cause of global irreversible blindness.25 Currently there are no effective remedies for POAG. MYOC encoding myocilin creates causal mutations and results in elevated intraocular pressure, which in turn causes glaucoma. Gene therapy that cultures Human Trabecular meshwork (HTM) cells encourages production of mutant myocilin, and therefore normalizes cell morphology and halting cell mortality. RNAi is used in eliminating malformed myocilin HTM cells as a gene silencing method. The first study to demonstrate that gene therapy can be used in silencing mutant MYOC was performed in 2009 by Li et al.25 Previous studies were aimed at introducing replacement cells to replace the damaged cells. MYOC-null individuals have remained healthy after the therapy. Additionally the therapy is capable of suppressing myosin without

F.I. Al-Saikhan the need to replace damaged cells; hence, it is economical and can be used in the treatment of other disorders.

Conclusion Research into the potential application of gene therapy in the treatment of various ophthalmic conditions produced encouraging results. Many of the studies to date have utilized animal models. The use of AVV vectors has facilitated the transfer of normal genes into animal cells for models with Leber’s Congenital Amaurosis, retinitis pigmentosa and Stargardt disease. Gene therapy for the treatment of red–green colorblindness has been successful in squirrel monkeys. Plans are at an advanced stage to start clinical trials. Researchers have also proved that CD59 can treat AMD. Gene therapy can be used to treat primary open angle glaucoma (POAG), and studies have confirmed the economic viable. It has been noted that these conditions are caused by mutations in specific genes which impair the production of essential proteins. The fundamental principle behind gene therapy in ophthalmology is that the injection of fully-functional genes into cells allows restoration of normal gene expression. Consequently, the required gene product will be produced thus preventing the escalation of a disease. However, the time requirement for human clinical trials results of gene therapy will take some time prior to routine use in patients.

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